The present invention relates to an assay method for assaying an analyte as a biological assay subject or for detecting the presence or absence thereof, which is useful for simple clinical diagnosis, and a kit and an assay device to be used for the method; more specifically, the present invention relates to an assay method for assaying a great number of combinations of one or more species of analytes contained in a fluid sample or the presence or absence thereof, and a kit and an assay device therefor.
For determining the disease affecting a patient in a laboratory test, several types of laboratory test results should collectively be examined. In general, patients should undergo several types of such tests for appropriate diagnosis and therapeutic treatment. However, one laboratory reagent can assay or detect one item in most cases in the prior art, so the sample volume drawn from a patient is increased in proportion to the number of tests, which works a physical burden on the patient.
Alternatively, it is required to carry out conventional immunological tests by using automatic assay devices, so the sample drawn from a patient is delivered to an institute equipped with such automatic assay devices, where the tests are conducted, and then, the test results are reported to the doctor. In such manner, the doctor can make diagnosis based on the results and the clinical conditions of the patient. Therefore, such device works as one cause of delay treatment because the doctor cannot make a decision instantly.
So as to overcome such problem, a method comprising a combination of immune reaction and chromatography (abbreviated as xe2x80x9cimmunochromatographyxe2x80x9d herein below) has been developed in recent years. The standard principle of conventional immunochromatography will now be described below.
In the assay device to be used for the conventional immunochromatography, the following zones are arranged; a loading zone for loading a fluid sample containing an analyte, at one end of a developing element in the form of porous sheet such as nitrocellulose film, a water absorption zone for receiving the fluid transferred through capillary action in the developing element, at the other end, a sealing zone containing a marker-labeled immune substance, located on a side close to the loading zone between the water absorption zone and the loading zone, and a detection zone where an immune substance to bind a complex composed of the analyte and the labeled substance is immobilized, the zone being arranged on a side apart from the loading zone.
By the assay method by using such assay device, a fluid sample containing an analyte to be assayed is firstly loaded on the loading zone, and the fluid sample is then transferred through capillary action to the sealing zone containing a marker-labeled immune substance. In the sealing zone, the marker-labeled immune substance and the analyte are bound together through immunological affinity, to form a marker-labeled immunocomplex. The marker-labeled immunocomplex is developed and transferred, through capillary action and/or diffusion in the developing element, to the detection zone, where an immune substance immobilized in the detection zone captures the complex. The marker in the marker-labeled immunocomplex captured in the detection zone is assayed or detected, whereby the amount or presence of the analyte contained in the fluid sample can be assayed.
Compared with enzyme immunoassay, another assay for immunochemical active substances, the method is characteristic in that no rinsing procedure is required during an intermediate stage of assaying and the assay can be done under naked eyes, essentially never requiring any device to detect the marker, and in that the reagent contained in the assay device is kept at dry state so it can be stored at ambient temperature for a long term. According to the conventional immunochromatography, a doctor can instantly examine a sample collected by himself, and hence, the doctor can inclusively take into account clinical conditions of a patient and the immunological test results of the patient, to diagnose the patient in a short time. Accordingly, the delay in the treatment will be less, advantageously.
A number of patent application have been laid open concerning immunochromatography. For example, the immunochromatography described in Japanese Patent Publication No. Hei 7-13640 is essentially the same as the prior art immunochromatography described above, characterized in that a ligand bound to an insoluble vesicle marker is used and the insoluble vesicle marker is colored liposome, colored polymer bead, or metal or polymer dye particle. However, the publication does not include any description about the simultaneous assay or detection of one or more species of biological substances such as antigen or antibody.
Japanese Patent No. 2504923 describes an immunochromatography essentially the same as the prior art immunochromatography, suggesting an analysis by a sandwich method wherein a complex captured in a detection zone is a marker-labeled receptor-analyte-receptor as well as simultaneous detection of a first analyte and a second analyte, having biological affinities different from each other. However, the publication does not suggest that the marker-labeled immunocomplex is captured through the complementary binding between the bases of nucleic acids in a detection zone or the applicability of the method to two or more analytes or the assay sensitivity thereof.
Alternatively, immunoassay methods can yield higher sensitivity when a large amount of immunochemically active substances can be immobilized, and in that case, the methods can detect the same levels of immunochemically active substances in a shorter time. Hence, a more highly sensitive assay technique in the field of immunochromatography has been desired.
It is thus an object of the present invention to provide an assay method useful for clinical diagnosis, which can simultaneously assay one or more species of biological substances or detect the presence or absence thereof, at a higher sensitivity, by means of a single assay device in a simple fashion, and a kit and an assay device for the assay.
A first aspect of the assay method of the present invention is an assay method by means of a kit, wherein a reagent and an assay device are separately arranged. The assay method is an assay method for assaying the amounts of one or more species of analytes present in a fluid sample or detecting the presence or absence thereof, comprising:
(1) putting a fluid sample containing one or more species of analytes in contact to a reagent containing one or more species of marker-labeled ligands each produced by binding a marker to a first ligand, and one or more species of bond element-labeled ligands each produced by binding a bond element consisting of nucleic acids with a predetermined base sequence depending on the analyte species, to a second ligand, to generate one or more species of specific complexes each composed of a specific analyte species, a specific marker-labeled ligand species specifically binding to the specific analyte species, and a specific bond element-labeled ligand species specifically binding to the specific analyte species;
(2) developing one or more species of generated complexes through capillary action in a developing element in a sheet form;
(3) capturing a complex depending on the analyte species, through the complementary binding between the bond element and an anti-bond element, in the detection zone produced by immobilizing independently anti-bond elements consisting of nucleic acids each having a complementary sequence to the base sequence of one bond element species in the complexes, thereby forming an independent band; and
(4) assaying or detecting the marker formed in the band in the detection zone.
Another embodiment of the assay method of the present invention is a method using an assay device integrally containing a reagent. The assay method is an assay method for assaying the amounts of one or more species of analytes present in a fluid sample or detecting the presence or absence thereof, comprising:
(1) loading a fluid sample containing one or more species of analytes on a developing element in a sheet form, thereby developing the fluid sample through capillary action in the developing element;
(2) transferring the fluid sample to put the sample in contact to a sealing zone sealing therein reagent components including one or more species of marker-labeled ligands each produced by binding a marker to a first ligand specifically reactive to a specific analyte species, and one or more species of bond element-labeled ligands each produced by binding a bond element consisting of nucleic acids with a predetermined base sequence depending on the analyte species, to a second ligand specifically reactive to the specific analyte species;
(3) developing one or more species of specific complexes each composed of a specific analyte species, a specific marker-labeled ligand species specifically binding to the specific analyte species, and a specific bond element-labeled ligand species specifically binding to the specific analyte species, or developing a reaction product under way of formation, through capillary action in the developing element;
(4) capturing complex depending on the analyte species through complementary binding between the bond element and an anti-bond element and forming an independent band therefor in a detection zone where each anti-bond element species having the complementary base sequence to the sequence of one bond element species in the complex is immobilized; and
(5) assaying or detecting the marker contained in the band formed in the detection zone.
The assay kit of the present invention is an assay kit for assaying one or more species of analytes in a sample or detecting the presence or absence thereof in a sample, the assay kit comprising a reagent and an assay device of a separate type from the reagent, wherein the reagent includes one or more species of marker-labeled ligands each produced by binding a marker to a first ligand specifically reactive to a specific analyte species, and one or more species of bond element-labeled ligands each produced by binding a bond element consisting of nucleic acids with a predetermined base sequence depending on the specific analyte species, to a second ligand specifically reactive to the specific analyte species; and
wherein the assay device includes a developing element in a sheet form, the developing element can develop analytes, reagent and analytes bound to the reagent through capillary action, and one or more species of anti-bond elements comprising a nucleic acid with a base sequence complementary to a bond element contained in the separate reagent are independently each kind immobilized in the detection zone of the developing element, whereby a complex of each analyte species is captured through the complementary binding between the bond element and an anti-bond element in the detection zone, thereby forming an independent band.
The assay device of the present invention characteristically is an assay device contained in the assay kit.
Furthermore, the assay device of the present invention is an assay device for assaying one or more species of analytes present in a sample or detecting the presence or absence thereof in the sample, wherein the assay device includes,
(1) a developing element in a sheet form, being capable of developing analytes, reagent and analytes bound to the reagent;
(2) a loading zone to receive a fluid sample from outside, the loading zone being positioned at one end of the developing element in a sheet form and capable of receiving a fluid sample from outside and having a sufficient feeding potency to transfer the received fluid sample to the other end to supply the fluid sample to be analyzed to a sealing zone sealing therein the reagent components;
(3) a sealing zone sealing therein reagent components including one or more species of marker-labeled ligands each produced by binding a marker to a first ligand specifically reactive to a specific analyte species, and one or more species of bond element-labeled ligands each produced by binding a bond element consisting of nucleic acids with a predetermined base sequence depending on the analyte species, to a second ligand specifically reactive to the specific analyte species, the sealing zone being arranged at a position close to the loading zone;
(4) a water absorption zone arranged at a position apart from the loading zone, the zone being capable of receiving the analytes, reagent and analytes bound to the reagent, after diffusion in the developing element; and
(5) a detection zone positioned between the sealing zone and the water absorption zone, where one or more species of anti-bond elements each with a base sequence complementary to one bond element are immobilized, whereby a complex formed from a marker-labeled ligand, an analyte species and a bond element-labeled ligand, depending on the analyte species, can be captured and detected.
In accordance with the present invention, one or more species of analytes can be assayed with a single kit or assay device at a high sensitivity.
In accordance with the present invention, the term xe2x80x9cligandxe2x80x9d means a molecule having a biological affinity with an analyte and being capable of specifically reacting with a specific analyte species, to form a pair. In accordance with the present invention, the term xe2x80x9cfirst ligandxe2x80x9d and xe2x80x9csecond ligandxe2x80x9d may have the same properties or may not have the same properties. If an analyte is an antigen, the first ligand and the second ligand may be antibodies; if an analyte is an antibody, the first ligand and the second ligand may be antigens. Another example of the combination of the analyte and ligands includes a combination of a receptor and ligands capable of binding to the receptor, a combination of a nucleic acid and complementary nucleic acids capable of binding to nucleic acids, a combination of lectin and specific sugars capable of binding to lectin.
In accordance with the present invention, the term xe2x80x9cbond elementxe2x80x9d means a nucleic acid which does not bind to an analyte but has a reactivity different from those ligands. In accordance with the present invention, the term xe2x80x9canti-bond elementxe2x80x9d means a nucleic acid with a base sequence at least partially complementary to the base sequence of the xe2x80x9cbond elementxe2x80x9d, and the anti-bond element binds to the bond element in a complementary fashion. The combination of the xe2x80x9cbond elementxe2x80x9d and the xe2x80x9canti-bond elementxe2x80x9d includes nearly infinite numbers of combinations, depending on the base sequences of nucleic acid molecules composing the bond element and the anti-bond element. From the respect of the complementary binding between the bond element and the anti-bond element, individual base sequences may satisfactorily be partially complementary or completely complementary to each other. As the nucleic acids functioning as the bond element and anti-bond element, use may be made of DNA, RNA, oligonucleotide, and polynucleotide, preferably including an oligonucleotide of a length of 10 mer or more to 100 mer or less.
Directly or indirectly through a substance, the anti-bond elements are bound and immobilized on the developing element in a detection zone. For example, the anti-bond elements are directly immobilized on the developing element, by covalently bonding a nucleic acid as the anti-bond element, through a functional group introduced into 5xe2x80x2 or 3xe2x80x2 terminus of a nucleic acid or introduced into a base of a nucleic acid, to a functional group contained in an insoluble support as the developing element. Depending on the one or more species of analytes to be assayed, individually different base sequences are predetermined for the anti-bond elements and are immobilized separately from each other, in the form of zones in the detection zone.
Through biotin introduced into 5xe2x80x2 or 3xe2x80x2 terminus of a nucleic acid or through biotin introduced into a nucleotide composing a nucleic acid, nucleic acids as anti-bond elements are bound to avidins or streptoavidins preliminarily bound to an insoluble support as the developing element, whereby nucleic acids can be immobilized on the developing element indirectly. By binding a nucleic acid, through a functional group introduced into 5xe2x80x2 or 3xe2x80x2 terminus of nucleic acid or introduced into a base of the nucleic acid, to a protein and then binding the nucleic acid-bound protein to an insoluble support as the developing element, nucleic acids as an anti-bond element can indirectly be immobilized on the developing element.
As a reagent component to be used in accordance with the present invention, use may be made of xe2x80x9cmarker-labeled ligandxe2x80x9d produced by binding a marker to a first ligand, and xe2x80x9cbond element-labeled ligandxe2x80x9d produced by binding a bond element consisting of nucleic acids with a predetermined base sequence, depending on the analyte species, to a second ligand. Separately from the developing element, the reagent component can compose a kit to be used in combination with the developing element. Additionally, the reagent component may satisfactorily be retained at a dry state in the sealing zone of the developing element.
As a marker contained in the marker-labeled ligand, specifically, use may be made of enzymatically active molecules, digoxigenin, metal colloid, colored latex, colored liposome, nucleic acid, biotin, avidin, fluorescent substance, luminescent substance, radioisotope and the like. Herein, the meaning of the term xe2x80x9ccoloringxe2x80x9d is not limited to the deposition of a color which can be discriminated visually, but includes the deposition of fluorescent substances and luminescent substances.
In accordance with the present invention, the xe2x80x9cdeveloping elementxe2x80x9d is in the form of sheet, and can develop analytes, reagent and analytes bound to the reagent in a chromatographic fashion. Preferably as the developing element, use may be made of porous insoluble supports, more specifically including plastic porous supports, cellulose porous supports and inorganic porous supports; still more specifically, use may be made of cellulose, nitrocellulose, cellulose acetate, nylon, silica or derivatives thereof, all being porous. In the individual multiple zones formed on the developing element, different materials may be used or used in combination. In some case, one face of each of plural zones may be reinforced with the same material as the material on the other face or with a different material from the material on the other face. Additionally, these zones may generally be dry if not used for assay.